Prostate cancer initially depends on androgens for growth and initially responds to androgen ablation therapy. Nevertheless, the cancer ultimately becomes resistant to anti-androgens and disease progression occurs. However, the tumors retain androgen receptor (AR) and its downstream signaling pathway. Resistance may be due to several factors. First, anti-androgens may display partial agonist behavior, the basis of which may vary with the compound. Second, mutant ARs may arise during treatment, with reduced ligand specificity or ligand-independent activity. These mutant ARs may show differential resistance to particular antagonists. An overall hypothesis of this proposal is that mutant ARs are selected during treatment of prostate cancer with anti-androgens and allow progression of androgen-independent disease. Our specific hypotheses are that: 1) The commonly used antagonists, bicalutamide and flutamide, differ in their precise mechanism of action, and thus mutant ARs resistant to one may not have a similar advantage against the other. 2) Partial agonism may in some cases be due to interactions of hormone- and antagonist-bound receptors. This suggests that disease progression may vary dependent on antagonist. Understanding mechanisms of androgen resistance, and if it varies with antagonist, may lead to improved treatment regimens and novel targets for therapy. Aim I. To determine the molecular basis for differential partial agonism of bicalutamide vs. flutamide. We will assess interaction of antagonist-bound ARs with nonreceptor proteins, and interaction of agonist- and antagonist-bound ARs in mixed ligand dimers. This will be analyzed biochemically using in vitro assays, including co-precipitation with glutathione-S-transferase-fused proteins, and functionally in transfected cells, for wild type AR and mutant forms found in prostate cancer. Aim II. To test whether prostate cancer growth and progression is delayed by sequential or combined antagonists, and whether this correlates with AR variation, in three complementary mouse models of prostate cancer. This Aim will utilize two new, prostate cancer mouse models: the VCaP xenograft model of hormone-sensitive (but not dependent) prostate cancer derived from a participant in the SPORE warm autopsy program, and the h/mAR TRAMP model ofhumaized AR in the TRAMP background. VCaP and h/mAR TRAMP mice will be randomized to groups treated with bicalutamide, flutamide, both or neither. Tumor growth between treament arms will be compared and AR cDNAs will be scanned for mutations. Mutations will be tested for functional effect on AR in vitro. Aim III. To determine whether antiandrogen treatment differentially affects human disease progression and correlates with distinct AR mutations. AR sequences will be determined in samples from the rapid autopsy program, for patients treated with flutamide vs. bicalutamide, in comparison to hormone-naive Ulm patient samples. Mutations will be compared to those in mice, and any novel mutations will be introduced into vectors to test the functional effect on AR in vitro.